The surface of Pneumocystis carinii is coated with an abundant and highly immunogenic glycoprotein of molecular weight 120,000. The cDNA sequences of clones encoding this Major Surface Glycoprotein (MSG) of rat P. carinii have been previously determined in this laboratory. While strong similarity exists for deduced amino acid sequences of these MSG clones, there is also marked variation. This raises the possibility that antigenic variation may be occurring in P. carinii as a method of evading host response. We have inserted the cDNA encoding a full length MSG into a baculovirus expression vector and have immunized rats with the full length non-glycosylated protein which was obtained in high yield from infected insect cells. Rats immunized with the recombinant protein were subsequently shown to develop antibody responses to MSG, demonstrating the potential of recombinant MSG to serve as a vaccine. While multiple variants of MSG have been cloned from cDNA libraries which were constructed from the lungs of several infected rats, the pattern and levels of expression of these individual variants has remained unclear. We therefore produced in rabbits and mice anti- peptide antisera which were specific for epitopes (15 amino acid length) unique to specific MSG variants. Using purified P. carinii from single rats, we observed that both the cyst and trophic forms of P. carinii were positive in immunofluorescent microscopy. The level of expression observed for a specific epitope ranged between 2% and 50% of organisms exhibiting fluorescence. Immunofluorescent microscopy of frozen specimens of purified P. carinii isolated from NIH colony rats in the period of 1990 to 1993 revealed that the level of expression of these epitopes could vary with time. One epitope (R1) showed a marked decrease in expression during this time (50% in 1990 to 5% in 1993) while others showed no change. While the level of expression of RI in the NIH rats had dropped to 5% by 1993, the expression of this epitope in P. carinii isolated from a nearby commercial rodent colony remained at 50% in 1993, demonstrating geographic variability. To assess the expression pattern of these epitopes in situ, immunohistochemistry was performed using the epitope specific antisera on serial sections of fixed rat lung. These sections clearly showed a focal, exclusive expression pattern of individual epitopes, suggesting a clonal proliferation.